Photoaffinity labeling of lactate dehydrogenase by the bis-azido analog of NAD+:P1-N6-(4-azidophenylethyl)adenosine-P2-[4-(3-azidopyridinio)butyl] diphosphate.
Lactate dehydrogenase from pig heart is inactivated by the NAD+-analog P1-N6-(4-azidophenylethyl)adenosine-P2-[4-(3-azidopyridinio) butyl]diphosphate (6) upon irradiation with UV light of wavelengths in the range from 300 to 380 nm. The decrease in enzyme activity can be prevented by the addition of NAD+ and oxalate. The modified enzyme shows a reduced binding capacity for its coenzyme as compared to native lactate dehydrogenase. The amount of incorporated coenzyme is deduced from the ribose content of inactivated enzyme. Tryptic digestion of the modified protein and separation of the peptides by HPLC yields 5 ribose-containing fractions. One of them, fraction 66, is split by treatment with nucleotide pyrophosphatase into two subfractions, 63 and 58. Only subfraction 63 contains ribose. Whereas peptide 58 shows a UV absorption spectrum similar to that of 4-(3-aminopyridinio)-butyl phosphate (3). Amino acid analyses of the peptides indicate that the inactivator forms covalent bonds with different parts of the protein: Peptide 63 is characterized by a great portion of hydrophobic amino acids whereas peptide 58 shows a high degree of hydrophilicity.
Woenckhaus
C
C
Klinikum der Johann Wolfgang Goethe Universität, Frankfurt.
Filbrich
R
R
D000345:Affinity Labels / Q000378:metabolism*; D000818:Animals; D001386:Azides / Q000138:chemical synthesis / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D007202:Indicators and Reagents; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism*; D009206:Myocardium / Q000201:enzymology; D009243:NAD / Q000138:chemical synthesis / Q000378:metabolism* / Q000031:analogs & derivatives; D010446:Peptide Fragments / Q000302:isolation & purification; D013552:Swine
D000345:Affinity Labels; D001386:Azides; D007202:Indicators and Reagents; D010446:Peptide Fragments; D009243:NAD; C067459:P(1)-N(6)-(4-azidophenylethyl)adenosine-P(2)-(4-(3-azidopyridinio)butyl)diphosphate; D007770:L-Lactate Dehydrogenase